Researchers have identified three mechanisms of gene exchange in bacteria. Conjugation is the cell-contact-dependent plasmid exchange mediated by conjugative plasmids (1). Transduction is gene transfer mediated through bacteriophages (2). Transformation is the process whereby a cell takes up and expresses genes encoded by extracellular DNA (3). Natural transformation is a normal physiological process exhibited by a wide range of bacteria (4,5). Natural transformation is distinct from artificial transformation, which is a widely used technique in molecular biology for the induction of competence in cells by chemical, enzymatic, or physical means.
There is considerable indirect evidence which suggests that natural transformation may be a mechanism of gene transfer in aquatic environments (6). First, several marine bacterial isolates have been reported to be naturally transformable (7,8). Second, aquatic environments have been shown to contain an abundance of dissolved DNA which could potentially act as transforming DNA (9).
Microbial gene transfer mechanisms may have evolved as a means for bacteria to adapt to changing environments and may represent a normal function of bacteria in aquatic and terrestrial ecosystems. The use of genetically engineered microorganisms in the environment and the spread of antibiotic resistances resulting from the use of antibiotics in medicine and agriculture may result in what is termed "genetic pollution". Genetic pollution is the introduction of new genetic material or the transfer of genes in the environment resulting from or related to anthropogenic activities. In contrast to other forms of pollution, genetic pollution has the capability of self-propagation once it is established in a component (i.e., recipient) of an ecosystem.
The potential of natural transformation to occur in a aquatic environments has not been studied extensively. However, the issue is significant since the United States Environmental Protection Agency is currently involved in permitting biotechnology firms to use genetically engineered microorganisms in the environment for agricultural, bioremediative, and pest control purposes. To decide whether such genetically engineered microorganisms will be permitted for use, decisions are based upon the nature of the modified gene sequences contained in the cells to be released, the organism to be used and the potential for survival of the organism in the environment. In nearly all instances, the survival of the released organism is determined by some type of viable count, usually a plate count.
Another area of concern regarding gene pools capable of transformation to other cells is the issue of cell killing in waste water facilities. Waste water contains high levels of coliform bacteria and gene transfer by conjugation and transformation has been documented in such environments (10,11). Chemical disinfection treatments such as chlorination or use of other halogens typically reduce coliform and pathogenic cell counts to acceptably low levels, prior to release of the water into rivers and estuaries. If the genomes (portions of the chromosome or plasmids) survive these treatments either in intact cells or released as free DNA, transfer of genes to the ambient aquatic microbial population can occur. The potential for pathogenic traits and antibiotic resistances to be spread to aquatic bacteria such as Vibrio species, many of which can cause gastroenteritis or septicaemia could result in serious health problems (12,13).
The present invention provides a high frequency of transformation estuarine strain capable for use in a novel detection system for assaying samples, such as samples containing a potentially useful genetically engineered microorganism or a sample from a waste water facility for the propensity of plasmid transformation in the samples. This provides an indication of the safety of using the genetically engineered microorganism as well as the level of potentially infectious genomes that could survive waste water treatments.